1. Field of the Invention
The present invention relates to lytic reagent compositions, diluents and methods for use in measuring total hemoglobin concentration in a blood sample, either manually or automatically, and for use in combination with a simultaneous leukocyte counting or differential counting of leukocyte subpopulations.
2. Discussion of the Prior Art
The determination of total hemoglobin is indicative of the oxygen-carrying capacity of whole blood. An ability to measure hemoglobin (Hgb) in blood samples is an essential part of diagnostic analysis and is also important for monitoring responsiveness to therapies directed towards diseases which affect hemoglobin and to therapies which are directed towards other diseases but which may have adverse side effects on the hemoglobin level.
Leukocytes in the peripheral blood of normal subjects consist of five types, i.e., lymphocytes, monocytes, neutrophils, eosinophils and basophils. The latter three types of leukocytes are collectively referred to as granulocytes. Different types of leukocytes have different biological functionalities. Counting and differentiating different types of leukocytes in a blood sample provides valuable information for clinical diagnosis.
The classification and counting of leukocytes has most commonly been conducted by the differential counting method which is also referred to the manual method. Automatic blood analyzers are also commonly used for counting leukocytes, which employs a hemolytic reagent to lyse erythrocytes and prepares a sample only containing leukocytes. The sample mixture then is analyzed by impedance method. A more sophisticated apparatus has been developed that counts different types of leukocytes (differential counting) including lymphoid (lymphocyte) and myeloid (monocyte and granulocyte) populations (U.S. Pat. No. 4,286,963 to Ledis et al.). Leukocytes have also been further differentiated into three subpopulations, i.e., monocytes, lymphocytes and granulocytes (U.S. Pat. No. 4,485,185 to Ledis et al.). Ideally, one would like to be able to accomplish multiple diagnostic analyses such as hemoglobin measurement and counting the number of leukocytes or differential counting of leukocyte subpopulations in a single automated step.
Among the many well-known methods for hemoglobin determination, the cyanide hemoglobin method has been recommended as a standard by the International Committee for Standardization in Hematology. Modification of this method by Matsubara and Okuzono has led to its wide usage in clinical laboratories. In this method, the iron ion of heme group in all forms of hemoglobin of the red cells are oxidized to methemoglobin by potassium ferricyanide. The methemoglobin is then complexed with cyanide anion, which has a very high affinity to iron ion of the heme group, to form a cyanmethemoglobin chromogen. This extremely stable chromogen has a maximum absorption at 540 nm, which is measured manually by UV spectrometry.
Despite of the stable chromogens formed by the standard cyanmethemoglobin method and its modified automatic methods, however, because of the potassium cyanide used, the reagent waste has caused enormous environmental concern. In last ten years, a tremendous effort has been given to develop automated hemoglobin analysis methods without utilizing cyanide.
Oshiro et al, Clin. Biochem. 1583 (1982), teach the use of a reagent for hemoglobin analysis which comprises sodium laurylsulfate (SLS) and Triton X-100 (a nonionic surfactant) in a neutral pH (7.2). The SLS is used to lyse erythrocytes and is believed to further produce a SLS-hemoglobin complex which has a maximum absorption at 539 nm and a shoulder at 572 nm. The reaction completes within 5-10 minutes and the total hemoglobin measurement is quantitative. However, as later explained in U.S. Pat. No. 5,242,832 (to Sakata), it is not possible with Oshiro's method to analyze leukocytes simultaneously with hemoglobin measurement.
U.S. Pat. No. 5,242,832 (to Sakata) discloses a cyanide-free lysing reagent for counting leukocytes and measuring the hemoglobin concentration in blood samples. The lysing reagent comprises at least one first surfactant which is a quaternary ammonium salt, at least one second surfactant which includes cationic and amphoteric surfactants, and at least one hemoglobin stabilizer selected from the group including Tiron, 8-hydroxyquinoline, bipyridine, 1-10-phenanthroline, phenolic compounds, bisphenol, pyrazole and derivatives, second phenyl 5-pyrazolone and derivatives, phenyl 3-pyrazolone, and imidazole and its derivatives. Sakata teaches that fractionation of the leukocytes into two or three groups including an aggregate of lymphocytes, an aggregate of monocytes, eosinophils and basophils, and an aggregate of neutrophils can only be accomplished by using at least two suitable surfactants and by rigorously controlling the surfactant concentration. Sakata also teaches that the preferred pH range of the lysing reagent is from 5.0 to 8.0. If the pH value is 3.0 or less, damage to the leukocytes increases thus rendering measurement of leukocytes difficult, and if the pH is 9.0 or more, the stability of hemoglobin deteriorates over time.
U.S. Pat. No. 5,612,223 (to Kim) discloses a cyanide-free method and reagent for determining hemoglobin in a whole blood sample. The reagent comprises a ligand selected from the group consisting of imidazole and derivatives, N-hydroxyacetamide, H-hydroxylamine, pyridine, oxazole, thiazole, pyrazole, pyrimidine, purine, quinoline and isoquinoline, and a surfactant with a strong erythrolytic capability selected from the group consisting of lauryl dimethylamine oxide and octylphenoxy polyethoxyethanol. The analysis method is fast, less than 10 seconds. However, the reagent only performs under an extreme alkaline condition, pH from 11 to 14. In addition, no capability of counting leukocytes or differentiating leukocyte subpopulations is taught by Kim.
U.S. Pat. No. 4,853,338 (to Benezra et al.) teaches a method of determining hemoglobin concentration in a blood sample. The method comprises the steps of (1) combining a sample with a reagent composition including an ionic surfactant at a concentration of from about 2 to 4%, wherein the reagent composition has a pH of from 11.3 to about 13.7 and is free of ionic cyanide to form a reaction mixture; (2) measuring the absorbance. This method is used for measuring hemoglobin concentration only. No leukocyte counting or differentiation of leukocyte subpopulations is taught by Benezra et al.
U.S. Pat. No. 5,250,437 (to Toda et al.) teaches a single reagent for determination of hemoglobin and analysis of leukocytes. The reagent is free of cyanide and contains (1) a combination of at least one quaternary ammonium salt and another surfactant including quaternary ammonium salt, nonionic surfactant, pyridinium salt, amphoteric surfactant and a cationic surfactant having a formula of R.sub.1 R.sub.2 R.sub.3 N.sup.+ -CH.sub.2 phenyl wherein R.sub.1, R.sub.2 and R.sub.3 are alkyl groups; and (2) at least one oxidant capable of oxidizing heme in hemoglobin. The reagent is capable of dividing leukocytes into at least two fractions. This method requires denaturing hemoglobin and oxidation of heme group.
U.S. Pat. No. 4,529,705 (to Larsen) teaches a reagent for combined diluting and lysing a blood sample for use in electronic enumeration of leukocytes and determination of hemoglobin concentration. The reagent comprises an aqueous solution of: (1) a quaternary ammonium salt detergent for lysing red blood cells in the sample; (2) at least one salt of an anion selected from the group consisting of sulfate, carbonate, formate, and acetate for preventing aggregation of platelets in the blood sample; and (3) an alkali metal cyanide for conversion of hemoglobin to a chromogen. This method provides convenience of combined diluting and lysing a blood sample using a single reagent for enumerating leukocytes and measuring hemoglobin concentration. However, it requires the presence of cyanide for hemoglobin measurement.
As described above, current hemoglobin measurement reagents and methods either suffer from toxicity of the reagent, or lack of ability to accomplish multiple diagnostic analyses such as hemoglobin measurement and counting the number of leukocytes or differential counting of leukocyte subpopulations in a single automated step.
A need arises for a new cyanide-free hemoglobin measurement method and a multifunctional reagent which is capable to accomplish multiple diagnostic analyses in a single automated step.